Glucuronyl C5-epimerase (herein, “C5-epimerase”) catalyzes the conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) in the second polymer modification step of heparin/heparan sulfate (HS) synthesis. The epimerase involved in heparin/HS synthesis has an absolute requirement for N-sulfate at the nonreducing side of the target HexA, the formation of which is catalyzed by a N-Deacetylase-N-sulfotransferase (NDST) in the first (preceding) step of biosynthetic polymer modification. Also, the epimerase is inhibited by O-sulfate groups near its site of action, so O-sulfation steps later in the heparin biosynthetic pathway inhibit epimerization or back-epimerization. The reaction involves reversible abstraction and readdition of a proton at C5 of the target hexuronic acid, via carbanion intermediate, and is believed to involve two polyprotic basic amino acids (esp. Lys).
The C5-epimerase, like other enzymes involved in heparin/HS biosynthesis, appears to be membrane bound or associated in the Golgi. Interestingly, solubilized epimerase catalyzes both (reversible) reactions, but no back-epimerization is detectable from microsomal fractions. C5-epimerase active protein was first purified and characterized from liver (Campbell et al., J. Biol. Chem. 269:26953-26958 (1994)).
Campbell, P. et al., reported the purification of the D-glucuronyl C5-epimerase from bovine liver (Campbell et al., J. Biol. Chem. 269: 26953-26958 (1994)), and several DNA sequences have also been reported. While the predicted size of the bovine C5-epimerase from genomic and cDNA sequences is 70.1 KD (618 amino acids) (discussed below), the most purified native preparate extracted as above contained predominant species of 52 and 20 kDa, indicating that proteolytic cleavage (processing) may have occurred. Detection of activity in larger MW (200 kDa) fractions from size-exclusion chromatography indicated that aggregation or oligomerization may occur. The enzyme has a broad pH range (6.5-7.5) of activity, having an optimum 7.4. The enzyme does not have a metal ion or other cofactor requirement. Kinetic studies unexpectedly revealed that the Km increases with increasing enzyme concentration, probably relating to polymeric substrate and steric hindrance, and/or oligomerization of the epimerase molecules.
Recently, Lindahl, U. and Li, J-P., WO98/48006, purified the 52 kDa C5-epimerase from bovine liver and obtained a partial amino acid sequence. Primers were made against an internal sequence and used to amplify a sequence from a bovine liver cDNA preparation. The bovine liver sequence was used to screen a bovine lung cDNA library. A sequence having an open reading frame of 444 amino acids was found, which corresponded to a polypeptide of 49.9 kDa. It was stated that the enzyme previously isolated from bovine liver was a truncated form of the native protein. Total RNA from bovine liver, lung and mouse mastocytoma were analyzed by hybridization to the bovine lung epimerase cDNA clone. Both bovine liver and bovine lung gave identical results, with a dominant transcript of about 9 kb and a weak 5 kb band. The mouse mastocytoma RNA only showed the transcript at about 5 kb.
The report of the cloning of a cDNA encoding a C5-epimerase from bovine lung also appeared in Li et al. J. Biol. Chem. 272: 28158-28163 (1997). Li et al. cloned and expressed the bovine lung epimerase in a baculovirus/insect cell system, which first assigned activity to a cloned (recombinant) sequence. The active recombinant protein was not purified for definitive assignment.
C5-epimerase cDNA sequences from Drosophila (GenBank Accession Number AAF57373), C. elegans (GenBank Accession Number P46555) and Methanococcus (GenBank Accession Number U67555) have been reported.
The enzymatic activity of the recombinant bovine epimerase reported by Lindahl et al. was relatively low. However, attempts to express the bovine lung C5-epimerase, the sole cloned mammalian epimerase, in systems that might yield a better production failed. Expression in mammalian cells, Saccharomyces cerevisiae, and E. coli have been attempted. To date, there have been no reports of the successful production of a soluble, active C5-epimerase. Therefore, it has not been possible to expand the early baculovirus cell system results into other recombinant systems or to use conventional expression methods such as mammalian, yeast and bacterial systems for expression of this enzyme.
Thus, there remains a need in the art for a highly active C5-epimerase, and for methods for production of larger amounts of the same.